The intricate programs of mammalian cell differentiation ultimately target chromatin, formulating chromatin environments accessible to or deflective of the transcriptional machinery and thus conducive to distinct gene expression profiles. How these chromatin structures are first established to set the transcription program and how these established structures are then re-instated on newly replicated DNA during cell division is the crux of epigenetics. Based on preliminary findings obtained during the previous funding period, we will comprehensively explore two major modulators of epigenetic information that we have investigated extensively: PRC1 and PRC2, and a third potential epigenetic modulator, CTCF. We will continue our investigation of the molecular basis of their specificity in targeting discrete regions of the genome, the dynamics controlling their activity, and how their activities convey appropriate transcription outputs. Two mammalian complexes that comprise Polycomb Group proteins, PRC1 and PRC2 are recognized epigenetic conveyers of transcriptional repression. PRC1 transmits repression through catalysis of monoubiquitination of histone H2A at lysine 119 and chromatin compaction. Through our extensive biochemical analyses, we demonstrated that PRC1 embodies discrete yet heterogenous complexes. In aim 1, we expand on our preliminary results showing that the distinguishing proteins for some of the PRC1 subgroups either convert PRC1 into a transcriptional activator, as in the case of the neuronal-specific protein, AUTS2, or exhibit cell-type specificity and a possible novel repressive mechanism as in the case of FBRSL1 in cardiomyocytes, or modulate PRC1 recruitment to chromatin as in the case of motor neuron enriched YAF1. The underlying mechanistic basis and the outcome to specific gene expression will be studied using biochemical and genomic approaches, respectively and with mouse models in the case of AUTS2 and FBRSL1. In aim 2, we continue our extensive analyses of PRC2 that catalyses methylation of histone H3 at lysine 27, a modification of repressive chromatin. We explore parameters regulating PRC2 activity including post-translational modifications of its catalytic subunit Ezh2, and the negative effects of naturally occurring, dominant negative histone mutants using biochemical analyses and CRISPR technology. With the foundation of our studies of interactions of long noncoding RNA with both Ezh2 and the PRC2 associated protein Jarid2, we expand into the role of these interactions in mediating specificity in PRC2 recruitment to chromatin. In aim 3, we pursue our preliminary results of RNA-mediated CTCF multimerization and its possible role in CTCF- mediated regulation of chromatin boundaries within the HOX gene cluster using ChIP-seq, sequence capture Hi-C and ChIA-PET technologies.